Regulation of the Saccharomyces cerevisiae DNA Replication Protein Cdc6p by the Cyclin Dependent Kinases

Author: Elsasser, Suzanne

Year: 1999

Degree: Dissertation (Ph.D.)

Advisor: Campbell, Judith L.

Committee Members: Campbell, Judith L.; Deshaies, Raymond Joseph; Dunphy, William G.; Kennedy, Mary B.; Varshavsky, Alexander J.

Option: Molecular Biology; Biochemistry

DOI: 10.7907/g19n-2f30

Abstract

A key feature of DNA replication in higher eukaryotes is that duplication of the genome occurs by DNA replication beginning at sequence-specific sites on the chromatin, and proceeds in each direction from this site of initiation. The DNA replication forks migrate toward each other but do not overlap. Furthermore, origin firing is restricted to one phase per cell cycle, and the cell regulates this by only allowing origin priming within a small window of the cell cycle. DNA replication, and cell cycle transitions in general, are governed by the cyclin dependent kinases, which are comprised of various cyclins associated with Cdk1p. The regulation of the Saccharomyces cerevisiae DNA replication protein Cdc6p by the cyclin dependent kinases is the subject of this thesis.

During the purification of Cdc6p from Saccharomyces cerevisiae, the Cdk1p was found to form a tight complex with Cdc6p. Since Cdc6p had previously been shown to be required for the initiation of DNA replication, and a subset of the cyclin dependent kinases had also been shown to modulate this event, this interaction was characterized. Cdc6p was found to interact with and be phosphorylated only by Cdk1p complexes associated with B-type cyclins. The B-type cyclins have been shown to have two roles in the initiation of DNA replication: one is triggering the firing of DNA replication origins at the G1/S transitions, and the other is blocking the subsequent priming of origins until the beginning of the next cell cycle. The interaction between Cdc6p and Cdk1p was shown to have relevance to the later mechanism of control. At the G1/S transition, Cdc6p is abruptly degraded, and the targeted degradation of Cdc6p is thought to be a key mechanism by which the cell closes the window of opportunity for origin priming. By extensive mutagenesis, phosphorylation of Cdc6p at two clusters of sites was shown to be required for this cell cycle dependent degradation. Degradation is furthermore mediated by ubiquitination, as was demonstrated by in vitro reactions. Genetic experiments suggest that stabilizing Cdc6p may lead to unscheduled initiation of replication.

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